Plant-produced glycoprotein comprising human-type sugar chain

ABSTRACT

The present invention provides a method for manufacturing a glycoprotein having a human-type sugar chain comprising a step in which transformed plant cell is obtained by introducing to a plant cell the gene of glycosyltransfetase and the gene of an exogenous glycoprotein, and a step in which the obtained transformed plant cell is cultivated.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.12/795,316 filed Jun. 7, 2010, now allowed, which is a continuation ofU.S. application Ser. No. 11/810,567, filed on Jun. 6, 2007, nowabandoned, which is a continuation of U.S. application Ser. No.10/870,635, filed on Jun. 17, 2004, now U.S. Pat. No. 7,388,081, whichis a divisional application of U.S. application Ser. No. 09/857,651,filed on Aug. 27, 2001, now U.S. Pat. No. 6,998,267, which is a nationalstage filing under 35 U.S.C. 371 of International ApplicationPCT/JP99/06881 filed Dec. 8, 1999, which claims priority to JapaneseApplication No. 10-350584, filed Dec. 9, 1998. The entire contents ofeach of the prior applications are incorporated by reference herein.

TECHNICAL FIELD

The present invention relates to expression of exogenous glycoproteinsby plants.

BACKGROUND ART

Many of the functional proteins in living organisms are glycoproteins.It has been elucidated that the diversity of the sugar chains inglycoproteins play several important roles physiologically (Lain, R. A.,Glycobiology, 4, 759-767, 1994).

In recent years, it has also become clear that the action of sugarchains can be divided into two categories. In the first case, sugarchains have a direct function as ligands for binding cells, or asreceptors for bacteria and viruses, in the clearance of glycoproteinsfrom the blood, lysosome targeting of lysosome enzymes and the targetingby glycoproteins toward specific tissues and organs. For example, thecontribution of glycoprotein sugar chains in the infection of targetcells by the AIDS virus (HIV) has been established (Rahebi, L. et al.Glycoconj. J., 12, 7-16, 1995). The surface of HIV is covered withenvelope protein gp120. The binding of gp120 sugar chains to the CD4 oftarget cells is the beginning of infection by the HIV virus. In thesecond case, the sugar chain itself is not the functional molecule butindirectly contributes to the formation of the higher-order structure ofproteins, solubility of proteins, protease resistance of proteins,inhibition of antigenicity, protein function modification, proteinregeneration rate adjustment, and adjustment of the amount of proteinsexpressed in cell layers. For example, sugar chains are instrumental inthe adjustment of the adhesion of nerve cell adhesion molecules whichare distributed widely in the nervous system (Edelman, G. M., Ann. Rev.Biochem., 54, 135-169, 1985).

In eukaryotes, glycoprotein sugar chains are synthesized on lipids ofthe Endoplasmic reticulum as precursor sugar chains. The sugar chainportion is transferred to the protein, then some of the sugar residueson the protein are removed in the Endoplasmic reticulum, and then theglycoprotein is transported to Golgi bodies. In the Goldi bodies, afterthe excess sugar residues have been removed, further sugar residues(e.g. mannose) are added and the sugar chain is extended (Narimatsu, H.,Microbiol. Immunol., 38, 489-504, 1994).

More specifically, for example, Glc3Man9GlcNAc2 on dolichol anchors istransferred to protein in the ER membrane (Moremen K. W., Trimble, R. B.and Herscovics A., Glycobiology 1994 April; 4 (2):113-25. Glycosidasesof the asparagine-linked oligosaccharide processing pathway; and Sturm,A. 1995 N-Glycosylation of plant proteins. In: New ComprehensiveBiochemistry. Glycoproteins, Vol. 29a., Montreuil, J., Schachter, H. andVliegenthart, J. F. G. (eds). Elsevier Science Publishers B. V., TheNetherlands, pp. 521-541). ER-glucosidase I and II removes three glucoseunits (Sturm, A. 1995, supra; and Kaushal G. P. and Elbein A. D., 1989,Glycoprotein processing enzymes in plants. In Methods Enzymology 179,Complex Carbohydrates Part F. Ginsburg V. (ed), Academic Press, Inc. NY,pp. 452-475). The resulting high mannose structure (Man9GlcNAc2) istrimmed by ER-mannosidase (Moremen K. W. et al, supra.; and Kornfeld, R.and Kornfeld, S., Annu. Rev. Biochem. 54, 631-664, 1985; Assembly ofasparagine-linked oligosaccharides). The number of mannose residuesremoved varies according to the differences in the accessibility to theprocessing enzymes. The isomers Man8-, Man7-, Man6- and Man5GlcNAc2 areproduced during processing by ER-mannosidase and Mannosidase I(Kornfeld, R. and Kornfeld, S., supra). When four mannose residues arecompletely removed by Mannosidase I (Man I), the product is Man5GlcNAc2.N-acetylglucosaminyl transferase I (GlcNAc I) transfersN-acetylglucosamine (GlcNAc) from UDP-GlcNAc to Man5GlcNAc2, resultingin GlcNAcMan5GlcNAc2 (Schachter, H., Narasimhan, S., Gleeson, P. andVella, G., Glycosyltransferases involved in elongation ofN-glycosidically linked oligosaccharides of the complex orN-acetylgalactosamine type. In: Methods Enzymol 98: Biomembranes Part L.Fleischer, S., and Fleischer, B. (ed), Academic Press, Inc. NY, pp.98-134 pp. 98-134, 1983). Mannosidase II (Man II) removes two mannoseresidues from GlcNAcMan5GlcNAc2, yielding GlcNAcMan3GlcNAc2 (Kaushal, G.P. and Elbein, A. D., supra; and Kornfeld, R. and Kornfeld, S., supra).The oligosaccharide GlcNAcMan4GlcNAc2 is used as a substrate ofN-acetylglucosaminyl transferase II (GlcNAc II) (Moremen K. W. et al,supra.; Kaushal, G. P. and Elbein, A. D., supra; and Kornfeld, R. andKornfeld, S., supra). FIG. 19 summarizes the above described structuresof N-linked glycans and enzymes involved in sugar chain modificationpathway in the Endoplasmic reticulum and Goldi bodies. In FIG. 19, ⋄denotes glucose, □ denotes GlcNAc, ◯ denotes mannose, ● denotesgalactose, and ▪ denotes sialic acid, respectively.

The sugar addition in the Golgi bodies is called terminal sugar chainsynthesis. The process differs widely among living organisms. The sugarchain synthesis depends on the type of eukaryote. The resulting sugarchain structure is species-specific, and reflects the evolution of sugaradding transferase and the Golgi bodies (Narimatsu, H., CellularBiology, 15, 802-810, 1996).

Regarding aspargine-linked (N-linked) sugar chains; in animals, thereare high mannose-type sugar chains, complex-type sugar chains andhybrid-type sugar chains. These structures are shown in FIG. 1. Thecomplex-type sugar chains in plants have α1,3 fucose and β1,2 xylosewhich are sugar residues that are not found in animals (Johnson, K. D.and Chrispeels, M. J., Plant Physiol., 84, 1301-1308, 1897, Kimura, Y.et al., Biosci. Biotech. Biochem., 56, 215-222, 1992). In the case ofN-linked sugar chains, sialic acid has been found in animal sugar chainsbut has not been found in plant sugar chains. Regarding galactose, whichis generally found in animal sugar chains, although the presence thereofhas been found in some plant sugar chains (Takahashi, N. and Hotta, T.,Biochemistry, 25, 388-395, 1986), the examples thereof are few. Thelinkage-type thereof is a β1,3 linkage (FEBS Lett 1997 Sep. 29, 415 (2),186-191, Identification of the human Lewis(a) carbohydrate motif in asecretory peroxidase from a plant cell suspension culture (Vacciniummytillus L.), Melo N S, Nimtz M, Contradt H S, Fevereiro P S, Costa J;Plant J. 1997 Dec. 12 (6), 1411-1417, N-glycans harboring the Lewis aepitope are expressed at the surface of plant cells, Fitchette-Laine AC, Gomord V, Cabanes M, Michalski J C, Saint Macary M, Foucher B,Cavelier B, Hawes C, Lerouge P, Faye L). This linkage is different fromthose found in animals.

Glycoproteins derived from humans include human erythropoietin (EPO). Inorder to produce glycoproteins with sugar chain structures similar tohumans, these glycoproteins are produced in animal host cells. However,EPO produced in animal cells has a sugar chain structure that isdifferent from the natural human sugar chain structure. As a result, invivo activity of EPO is reduced (Takeuchi, M. et al., Proc. Natl. Acad.Sci. USA, 86, 7819-7822, 1989). The sugar chain structure in otherproteins derived from humans, such as hormones and interferon, have alsobeen analyzed and manufactured with the same glycosylation limitations.

The methods used to introduce exogenous genes to plants include theAgrobacterium method (Weising, K. et al., Annu. Rev. Genet., 22, 421,1988), the electroporation method (Toriyama, K. et al., Bio/Technology,6, 1072, 1988), and the gold particle method (Gasser, C. G. and Fraley,R. T., Science, 244, 1293, 1989). Albumin (Sijmons, P. C. et al.,Bio/Technology, 8, 217, 1990), enkephalin (Vandekerckhove, J. et al.,Bio/Technology, 7, 929, 1989), and monoclonal antibodies (Benvenulo, E.et al., Plant Mol. Biol., 17, 865, 1991 and Hiatt, A. et al., Nature,342, 76, 1989) have been manufactured in plants. Hepatitis B virussurface antigens (HBsAg) (Mason, H. S. et al., Proc. Natl. Acad. Sci.USA., 89, 11745, 1992) and secretion-type IgA (Hiatt, A. and Ma, J. S.K., FEBS Lett., 307, 71, 1992) have also been manufactured in plantcells. However, when human-derived glycoproteins are expressed inplants, the sugar chains in the manufactured glycoproteins havedifferent structures than the sugar chains in the glycoproteins producedin humans because the sugar adding mechanism in plants is different fromthe sugar adding mechanism in animals. As a result, glycoproteins do nothave the original physiological activity and may be immunogenic inhumans (Wilson, I. B. H. et al., Glycobiol., Vol. 8, No. 7, pp. 651-661,1998).

DISCLOSURE OF THE INVENTION

The purpose of the present invention is to solve the problems associatedwith the prior art by providing plant-produced recombinant glycoproteinswith mammalian, e.g., human-type sugar chains.

The present invention is a method of manufacturing a glycoprotein havinga human-type sugar chain comprising a step in which a transformed plantcell is obtained by introducing to a plant cell the gene of an enzymecapable of conducting a transfer reaction of a galactose residue to anon-reducing terminal acetylglucosamine residue and the gene of aexogenous glycoprotein, and a step in which the obtained transformedplant cell is cultivated.

In the present invention, the glycoprotein with a human-type sugar chaincan comprise a core sugar chain and an outer sugar chain, the core sugarchain consists essentially of a plurality of mannose andacetylglucosamine, and the outer sugar chain contains a terminal sugarchain portion with a non-reducing terminal galactose.

In the present invention, the outer sugar chain can have a straightchain configuration or a branched configuration. In the presentinvention, the branched sugar chain portion can have a mono-, bi-, tri-or tetra configuration. In the present invention, the glycoprotein cancontain neither fucose nor xylose.

The present invention is also a plant cell having a sugar chain addingmechanism which can conduct a transfer reaction of a galactose residueto a non-reducing terminal acetylglucosamine residue, wherein the sugarchain adding mechanism acts on a sugar chain containing a core sugarchain and an outer sugar chain, wherein the core sugar chain consistsessentially of a plurality of mannose and acetylglucosamine, and whereinthe outer sugar chain contains a terminal sugar chain portion with anon-reducing terminal galactose.

In the present invention, a glycoprotein with a human-type sugar chainis obtained using this method.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1. A schematic drawing of typical N-linked sugar chainconfigurations.

FIG. 2. Schematic drawings of the cloning method for hGT. A: a schematicillustration showing the amplification of a hGT gene. B: a schematicillustration showing the cloning of the hGT gene into plasmid pBlue SK⁺.

FIG. 3. Schematic drawings of the method used to construct vector pGAhGTfor hGT expression.

FIG. 4. A photograph showing a Southern blot analysis of a genome ofcultivated transformed tobacco cells. FIG. 4 (A) shows electrophoresisafter the genome DNA (40 μg) has been digested by EcoRI and HindIII. Thenumbers at the left indicate the position of the DNA molecular weightmarker. FIG. 4 (B) shows a schematic drawing of a 2.2 kb fragmentcontaining a promoter, hGT and terminator, which is integrated into thetransformed cell.

FIG. 5. FIG. 5 is a photograph of the Western blotting of immunoreactiveprotein from transformed tobacco BY2 cells (WT) and wild type tobaccoBY2 cells (WT). The protein was denatured, electrophoresed on 10%SDS-PAGE, and then transferred electrically to nitrocellulose film. Thesamples were as follows: lane 1=GT1 cell extract; lane 2=GT 6 cellextract; lane 3=GT8 cell extract; lane 4=GT9 cell extract; lane 5=wildtype cell extract; lane 6=GT1 microsome fragment; lane 7=GT6 microsomefragment; lane 8=GT8 microsome fragment; lane 9=GT9 microsome fragment;lane 10=wild type microsome fragment.

FIG. 6. An electrophoresis photograph showing the detection ofgalactosylated glycoprotein using Ricinus communis (RCA₁₂₀) affinitychromatography. The electrophoresed gel was visualized by silverstaining. Lanes 1 and 2 show the protein from wild type BY2 cells, whileLanes 3 and 4 show the protein from transformed GT6 cells. The molecularweight is in KDa units.

FIG. 7. A photograph of Western blotting a showing the detection ofgalactosylated glycoprotein using Ricinus communis (RCA₁₂₀) affinitychromatography. After the electrophoresed gel had been blotted on anitrocellulose membrane, this membrane was visualized by lectin (RCA₁₂₀)staining. Lanes 1 and 2 show the protein from a wild type BY2 cell,while Lanes 3 and 4 show the protein from transformed GT6 cells. Themolecular weight is in KDa.

FIG. 8. A photograph of a blotting in which the galactosylatedglycoprotein from Ricinus communis (RCA₁₂₀) affinity chromatography wasprobed with an antiserum specific to xylose in complex-type plantglycans. Lanes 1 and 2 show the total protein extracts from BY2 and GT6,respectively, and Lane 3 shows the glycoprotein from GT6 after RCA₁₂₀affinity chromatography. The molecular weight is in KDa units.

FIG. 9. A schematic drawing of a plasmid pBIHm-HRP which is a binaryvector with a kanamycin-resistant gene and a hygromycin-resistant gene,and has a HRP cDNA.

FIG. 10. Photographs of isoelectric focusing and Western blotting whichshow HRP production in a suspension culture of transgenic cells. FIG. 10(A) shows the results of isoelectric focusing and FIG. 10 (B) shows theresults of Western blotting. The abbreviations are as follows:WT=wild-type; BY2-HRP 1, 5 and 7=the clone numbers for BY2 cellstransformed with a HRP gene; and GT-6-HRP 4, 5 and 8=the clone numbersfor GT6 cells transformed with a HRP gene.

FIG. 11. A graph showing the reverse-phase HPLC pattern of a PA sugarchain eluted in 0-15% acetonitrile linear gradient in 0.02% TFA over 60minutes and at a flow rate of 1.2 ml/min. I-XI shows the fractionseluted and purified from size-fractionation HPLC. Excitation wavelengthand emission wavelength were 310 mm and 380 mm, respectively.

FIG. 12. Graphs showing the size-fractionation HPLC pattern of the PAsugar chain in FIG. 11. Elution was performed in a 30-50% water gradientin the water-acetonitrile mixture over 40 minutes and at a flow rate of0.8 ml/min. The excitation wavelength and emission wavelength were 310nm and 380 nm, respectively.

FIG. 13. A graph showing the elution position of peak-K2 on reversephase HPLC wherein two standard sugar chain products A and B arecompared with the peak K2. The elution conditions were the same as inFIG. 11. That is, elution was performed in 0-15% acetonitrile lineargradient in 0.02% TFA over 60 minutes and at a flow rate of 1.2 ml/min.A and B show the structures of products A and B.

FIG. 14. Graphs showing the SF-HPLC profiles of galactosylated PA sugarchains obtained after exoglycosidase digestion. Elution was performed ina 30-50% water gradient in the water-acetonitrile mixture over 25minutes and at a flow rate of 0.8 ml/min. (A) PA-sugar chain K-2: I isthe elution position of the galactosylated PA sugar chain used; II isβ-galactosidase digests of I; III is a N-acetyl-β-D-glucosaminidasedigests of II; IV is jack bean α-mannosidase digests of III. (B)PA-sugar chain L: I is the elution position of the galactosylated PAsugar chain used; II is β-galactosidase digests of I; III isN-acetyl-β-D-glucosaminidase digests of II; IV is α1,2 mannosidasedigests of III; V is jack bean α-mannosidase digests of III.

FIG. 15. Estimated structures of the N-linked glycans obtained from thetransformed cells. The numbers in the parentheses indicate the molarratio.

FIG. 16. Photographs of Ricinus communis 120 agglutinin (RCA₁₂₀)affinity chromatography showing the detection of glycosylated HRP. FIG.16 (A) shows the results from silver staining, and FIG. 16 (B) shows theresults from lectin RCA₁₂₀ staining. The lectin-stained filter was cutinto strips and then probed using lectin RCA₁₂₀ pre-incubated withbuffer alone (I and II) or incubated in buffer with excess galactose(III). In (II), HRP was treated with β-galactosidase from Diplococcuspneumoniae before SDS-PAGE. Lane 1 is a collected fraction containingBY2-HRP and Lane 2 is a collected fraction containing GT6-HRP. Thenumbers to the left refer to the location and the size (KDa) of thestandard protein.

FIG. 17. A graph showing the results of reverse-phase HPLC of the PAsugar chains from purified HRP after RCA₁₂₀ affinity chromatography.

FIG. 18. Photographs of Western blotting showing immune detection ofplant specific complex-type glycans. The purified HRP is fractioned bySDS-PAGE, transferred to nitrocellulose, and confirmed with rabbitanti-HRP (A) and an antiserum which is specific for complex-type glycansof plants (B). Lane 1=galactosylated HRP from GT6-HRP after RCA₁₂₀affinity chromatography; Lane 2=purified HRP from BY2-HRP. The positionof the molecule size marker is shown to the left in KDa. Thegalactosylated N-glycan on HRP derived from the transformant GT6-HRPcells did not react with an antiserum which has been shown tospecifically react with β1,2 xylose residue indicative of plantN-glycans.

FIG. 19. Structures of N-linked glycans and enzymes involved in thesugar chain modification pathway in Endoplasmic reticulum and Goldibodies. ⋄ denotes glucose, □ denotes GlcNAc, ◯ denotes mannose, ●denotes galactose, and ▪ denotes sialic acid, respectively.

FIG. 20. Structures of N-linked glycans and the ratio of each N-linkedglycan in GT6 cell line along with those in wild-type BY2 cell linedetermined similarly. □ denotes GlcNAc, ◯ denotes mannose, ● denotesgalactose, and ▪ denotes sialic acid, respectively.

FIG. 21 illustrates one of the embodiment of the present invention. InGT6 cell line, the isomers Man7-, Man6- and Man5GlcNAc2 were observed.Because those high-mannose type oligosaccharides will be converted bysome glycan processing enzymes to be substrates forβ1,4-galactosyltransferase (Gal T), introduction of GlcNAc I, Man I andMan II cDNAs could more efficiently lead the oligosaccharideMan7-5GlcNAc2 to GlcNAcMan3GlcNAc2, which can be a substrate of GalT.

FIG. 22 also illustrates another the embodiment of the presentinvention. 1,4-Galactosyltransferase (Gal T) uses UDP-galactose as adonor substrate and GlcNAc2Man3GlcNAc2 as an acceptor substrate.Efficient supply of UDP-galactose will enhance the Gal T enzyme reactionand more galactosylated oligosaccharide will be produced.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described in further detail.In performing the present invention, unless otherwise indicated, anyconventional technique can be used. These include methods for isolatingand analyzing proteins as well as immunological methods. These methodscan be conducted by using commercial kits, antibodies and markers.

The method of the present invention relates to a method of manufacturingglycoproteins with human-type sugar chains. In this specification,“human-type sugar chain” refers to a sugar chain with a galactoseresidue linked to a N-acetylglucosamine residue. The galactose residuein the human-type sugar chain can be the terminal sugar chain or asialic acid residue can be linked to the outside of the galactoseresidue. Preferably, the glycoprotein of the present invention at leasthas no xylose or fucose linked to one or more of the following portions:the core sugar chain portion, the branched sugar chain portion, or theterminal sugar chain portion of the human-type sugar chain. Morepreferably, neither xylose or fucose should be linked to any portion ofthe human-type sugar chain, and ideally there should be no xylose orfucose contained in the human-type sugar chain at all.

The plant cells can be any plant cells desired. The plant cells can becultured cells, cells in cultured tissue or cultured organs, or cells ina plant. Preferably, the plant cells should be cultured cells, or cellsin cultured tissue or cultured organs. Most preferably, the plant cellsshould be cells in whole plants, or portions thereof, that produceglycoproteins with human-type sugar chains. The type of plant used inthe manufacturing method of the present invention can be any type ofplant that is used in gene transference. Examples of types of plantsthat can be used in the manufacturing method of the present inventioninclude plants in the families of Solanaceae, Poaeae, Brassicaceae,Rosaceae, Leguminosae, Curcurbitaceae, Lamiaceae, Liliaceae,Chenopddiaceae and Umbelliferae.

Examples of plants in the Solanaceae family include plants in theNicotiana, Solanum, Datura, Lycopersicon and Petunia genera. Specificexamples include tobacco, eggplant, potato, tomato, chili pepper, andpetunia.

Examples of plants in the Poaeae family include plants in the Oryza,Hordenum, Secale, Saccharum, Echinochloa and Zea genera. Specificexamples include rice, barley, rye, Echinochloa crus-galli, sorghum, andmaize.

Examples of plants in the Brassicaceae family include plants in theRaphanus, Brassica, Arabidopsis, Wasabia, and Capsella genera. Specificexamples include Japanese white radish, rapeseed, Arabidopsis thaliana,Japanese horseradish, and Capsella bursa-pastoris.

Examples of plants in the Rosaceae family include plants in the Orunus,Malus, Pynus, Fragaria, and Rosa genera. Specific examples include plum,peach, apple, pear, Dutch strawberry, and rose.

Examples of plants in the Leguminosae family include plants in theGlycine, Vigna, Phaseolus, Pisum, Vicia, Arachis, Trifolium, Alfalfa,and Medicago genera. Specific examples include soybean, adzuki bean,kidney beans, peas, fava beans, peanuts, clover, and alfalfa.

Examples of plants in the Curcurbitaceae family include plants in theLuffa, Curcurbita, and Cucumis genera. Specific examples include gourd,pumpkin, cucumber, and melon.

Examples of plants in the Lamiaceae family include plants in theLavandula, Mentha, and Perilla genera. Specific examples includelavender, peppermint, and beefsteak plant.

Examples of plants in the Liliaceae family include plants in the Allium,Lillum, and Tulipa genera. Specific examples include onion, garlic,lily, and tulip.

Examples of plants in the Chenopodiaceae family include plants in theSpinacia genera. A specific example is spinach.

Examples of plants in the Umbelliferae family include plants in theAngelica, Daucus, Cryptotaenia, and Apitum genera. Specific examplesinclude Japanese udo, carrot, honewort, and celery.

Preferably, the plants used in the manufacturing method of the presentinvention should be tobacco, tomato, potato, rice, maize, radish,soybean, peas, alfalfa or spinach. Ideally, the plants used in themanufacturing method of the present invention should be tobacco, tomato,potato, maize or soybean.

In this specification, “an enzyme capable of conducting a transferreaction of a galactose residue to a non-reducing terminalacetylglucosanine residue” refers to an enzyme capable of conducting atransfer reaction of a galactose residue to a non-reducing terminalacetylglucosamine residue produced when a sugar chain is added aftersynthesis of the protein portion of the glycoprotein in the plant cell.Specific examples of these enzymes include galactosyltransferase,galactosidase, and β-galactosidase. These enzymes can be derived fromany animal desired. Preferably, these enzymes should be derived from amammal, and ideally these enzymes should be derived from a human.

In this specification, “the gene of an enzyme capable of conducting atransfer reaction of a galactose residue to a non-reducing terminalacetylglucosamine residue” can be a gene which can be isolated from ananimal cell using a nucleotide sequence of an encoded enzyme well knownin the art, or commercially available genes altered for expression inplants.

In this specification, “gene” usually refers to the structural geneportion. A control sequence such as a promoter, operator and terminatorcan be linked to the gene so as to properly express the gene in a plant.

In this specification, “exogenous glycoproteins” refers to glycoproteinswhose expression in plants is the result of genetic engineeringmethodologies. Examples of these exogenous glycoproteins includeenzymes, hormones, cytokines, antibodies, vaccines, receptors and serumproteins. Examples of enzymes include horseradish peroxidase, kinase,glucocerebrosidase, α-galactosidase, tissue-type plasminogen activator(TPA), and HMG-CoA reductase. Examples of hormones and cytokines includeenkephalin, interferon alpha, GM-CSF, G-CSF, chorion stimulatinghormone, interleukin-2, interferon-beta, interferon-gamma,erythropoietin, vascular endothelial growth factor, humanchoriogonadotropin (HCG), leuteinizing hormone (LH), thyroid stimulatinghormone (TSH), prolactin, and ovary stimulating hormone. Examples ofantibodies include IgG and scFv. Examples of vaccines include antigenssuch as Hepatitis B surface antigen, rotavirus antigen, Escherichia colienterotoxin, malaria antigen, rabies virus G protein, and HIV virusglycoprotein (e.g., gp120). Examples of receptors and matrix proteinsinclude EGF receptors, fibronectin, a1-antitrypsin, and coagulationfactor VIII. Examples of serum proteins include albumin, complementproteins, plasminogen, corticosteroid-binding globulin, throxine-bindingglobulin, and protein C.

In this specification, “genes of exogeneous glycoproteins” refers to agene, which can be isolated from a cell using a nucleotide sequence ofan encoded protein well known in the art, or commercially availablegenes altered for expression in plants.

The gene of the enzymes capable of conducting a transfer reaction of agalactose residue to a non-reducing terminal acetylglucosamine residueand the genes of exogenous glycoproteins can be introduced to the plantcells using a method well known in the art. These genes can beintroduced separately or simultaneously. Examples of methods forintroducing genes to plant cells include the Agrobacterium method, theelectroporation method and the particle bombardment method.

Using any method well known in the art, the plant cells with introducedgenes can be tested to make sure the introduced genes are expressed.Examples of such methods include silver staining or augmentation,Western blotting, Northern hybridization, and enzyme activity detection.Cells that express the introduced genes are referred to as transformedcells.

Transformed cells, which express enzymes capable of conducting atransfer reaction of a galactose residue to a non-reducing terminalacetylglucosamine residue and exogenous glycoproteins, express exogenousglycoproteins with human-type sugar chains. In other words, thetransformed cells have human-type sugar chain adding mechanisms. Bycultivating these transformed cells, glycoproteins with human-type sugarchains can be mass produced. Human-type glycoproteins contain core sugarchains and outside sugar chains. The core sugar chains consistessentially of a plurality of mannose or acetylglucosamine. The outsidesugar chains in these glycoproteins contain non-reducing terminal sugarchain portions. The outside sugar chains can have a straight chainconfiguration or a branched chain configuration. The branched sugarchain portion has a mono-, bi-, tri- or tetra configuration. Theglycoproteins manufactured using these transformed cells ideally do notcontain any fucose or xylose.

These transformed plant cells can remain in a cultured state or can bedifferentiated into specific tissues or organs. Alternatively, they canalso be generated into plants. In this case, the transformed plant cellscan be present in the entire plant or in specific portions of the plant,such as seed, fruit, nut, leaf, root, stem or flower of the plant.

Glycoproteins with human-type sugar chains can be manufactured by thetransformed plant cells and then be isolated or extracted from the plantcells. The method for isolating the glycoproteins can be any method wellknown in the art. The glycoproteins of the present invention can be usedin foodstuffs while remaining inside the transformed cells, or theglycoproteins of the present invention can be administered to animalsincluding humans without antigenicity because of the added human-typesugar chains.

Hereinafter, the present invention will be described in detail by way ofillustrative, but not restrictive, examples.

Example 1 Cloning Human β1,4 Galactose Transferase Genes

β1,4 Galactosyltransferase (hGT) genes (EC2.4.1.38) have already beencloned. A primary configuration consisting of 400 amino acids has beendiscovered (Masri, K. A. et al., Biochem. Biophys. Res. Commun., 157,657-663, 1988).

(1) Primer Preparation and Template DNA

The following primers were prepared with reference to the report byMasri et al.

(Sequence ID: 1) hGT-5Eco: 5′-AAAGAATTCGCGATGCCAGGCGCGCGTCCCT-3′(Sequence I.D: 2) hGT-2Sal: 3′-TCGATCGCAAAACCATGTGCAGCTGATG-5′(Sequence I.D: 3) hGT-7Spe: 3′-ACGGGACTCCTCAGGGGCGATGATCATAA-5′(Sequence I.D: 4) hGT6Spe: 5′-AAGACTAGTGGGCCCCATGCTGATTGA-3′

Human genome DNA, human placenta cDNA, and human kidney cDNA purchasedfrom Clontech were used as the template DNA.

(2) Cloning the hGT Gene cDNA

(i) Human genome DNA was used as the template and hGT-5Eco and hGT-7Spewere used as the primers; (ii) Human placenta cDNA was used as thetemplate and hGT-2Sal and hGT6Spe were used as the primers. The two werecombined and a PCR reaction was performed under the followingconditions. Then, 0.4 kb and 0.8 kb fragments containing hGT encodedareas were obtained.

(PCR Reaction Mixture)

1 μl template DNA, 5 μml 10×PCR buffer solution, 4 μl dNTPs (200 mM),the primers (10 pmol), and 0.5 μl (Takara Shuzo Co., Ltd.) Tagpolymerase (or 0.2 μl Tub polymerase), water was added to make 50 μl.

(PCR Reaction Conditions)

First Stage: 1 cycle, denaturation (94° C.) 5 minutes, annealing (55°C.) 1 minute, extension (72° C.) 2 minutes. Second Stage: 30 cycles,denaturation (94° C.) 1 minute, annealing (55° C.) 1 minute, extension(72° C.) 2 minutes. Third Stage: 1 cycle, denaturation (94° C.) 1minute, annealing (55° C.) 2 minutes, extension (72° C.) 5 minutes.

The two fragments were combined to form hGT gene cDNA and cloned inpBluescript II SK+ (SK). The pBluescript II SK+ (SK) was purchased fromStratagene Co., Ltd. FIG. 2 shows the structure of a plasmid containinghGT gene cDNA. This shows Sequence No. 5 in the hGT gene nucleotidesequence and Sequence No. 6 in the estimated amino acid sequence. Thisnucleotide sequence differed from the hGT sequence published by Masri etal. (see above) in the following ways: a) The nucleotides are differentin that the A in Position No. 528 is G, the C in Position No. 562 is T,and the A in Position No. 1047 is G, however the encoded amino acidsequence is not changed; b) Nine nucleotides at positions from PositionNo. 622 to Position No. 630 are missing; c) The G in Position No. 405 isA and the T in Position No. 408 is A. These nucleotide changes were madeduring primer preparation such that the 0.4 kb fragment and 0.8 kbfragment are connected. There are two start codons (ATG) in hGT genecDNA. In this experiment, however, the gene is designed such thattranslation begins from the second initial codon (Position No. 37).

Example 2 Introduction of the hGT Gene to a Cultivated Tobacco Cell

(1) It has been reported that hGT is expressed in an active form inEscherichia coli (Aoki, D. et al., EMBO J., 9, 3171, 1990 and Nakazawa,K. et al., J. Biochem., 113, 747, 1993). In order for a cultivatedtobacco cell to express hGT, the expression vector pGAhGT had to bestructured as shown in FIG. 3. A cauliflower mosaic virus 35S promoter(CaMV 35S promoter), which drives gene expression constitutively inplant cells, was used as the promoter. A kanamycin-resistance gene wasused as the selection marker. The pGAhGT was introduced to thecultivated tobacco cell by means of Agrobacterium method.

The Agrobacterium method was performed using the triparental matingmethod of Bevan et al. (Bevan, M., Nucleic Acid Res., 12, 8711, 1984).Escherichia coli DH5α. (suE44, DlacU169, (φ80lacZDM15), hsdR17)(Bethesda Research Laboratories Inc.: Focus 8 (2), 9, 1986) withpGA-type plasmids (An, G., Methods Enzymol. 153, 292, 1987) andEscherichia coli HB101 with helper plasmid pRK2013 (Bevan, M., NucleicAcid Res., 12, 8711, 1984) were left standing overnight and 37° C. in a2×YT medium containing 12.5 mg/l tetracycline and 50 mg/l kanamycin, andAgrobacterium tumefaciens EHA101 was left standing over two nights at28° C. in a 2×YT medium containing 50 mg/l kanamycin and 25 mg/lchloramphenicol. Then, 1.5 ml of each cultured medium was removed andplaced into an EPPENDORF tube. After the cells of each strain werecollected, the cells were rinsed three times in an LB medium. The cellsobtained in this manner were then suspended in 100 μl of a 2×YT medium,mixed with three types of bacteria, applied to a 2×YT agar medium, andcultivated at 28° C. whereby the pGA-type plasmids, then underwentconjugal transfer from the E. coli to the Agrobacterium. Two days latersome of the colony appearing on the 2×YT agar plate was removed using aplatinum loop, and applied to an LB agar plate containing 50 mg/lkanamycin, 12.5 mg/l tetracycline, and 25 mg/l chloramphenicol. Aftercultivating the contents for two days at 28° C., a single colony wasselected.

Transformation of the cultivated tobacco cells was performed using themethod described in An, G., Plant Mol. Bio. Manual, A3, 1. First, 100 μlof Agrobacterium EHA101 with pGA-type plasmids cultivated for 36 hoursat 28° C. in an LB medium containing 12.5 mg/l tetracycline and 4 ml ofa suspension of cultivated tobacco cells Nicotiana tabacum L. cv. brightyellow 2 (Strain No. BY-2 obtained using Catalog No. RPC1 from the PlantCell Development Group of the Gene Bank at the Life Science TsukubaResearch Center), in their fourth day of cultivation, were mixedtogether thoroughly in a dish and allowed to stand in a dark place at25° C. Two days later, some of the solution was removed from the dishand the supernatant was separated out using a centrifuge (1000 rpm, 5minutes). The cell pellet was introduced to a new medium and centrifugedagain. The cells were innoculated onto a modified LS agar plate with150-200 mg/l kanamycin and 250 mg/l carbenicillin. This was allowed tostand in darkness at 25° C. After two to three weeks, the cells grown tothe callus stage were transferred to a new plate and clones wereselected. After two to three weeks, the clones were transferred to a 30ml modified LS medium with kanamycin and carbenicillin. This selectionprocess was repeated over about one month. Six resistant strains wererandomly selected from the resistant strains obtained in this manner (GT1, 4, 5, 6, 8 and 9).

(2) Verification of the Introduced hGT Genes

In the resistant strains, a 2.2 kb fragment containing a CaMV35Spromoter and an hGT gene cDNA-NOS terminator in the T-DNA was confirmedin the genomic DNA of the cultivated tobacco cells using a Southern blotanalysis. The Southern method was performed after the genomic DNA hadbeen prepared from the resistant strains mentioned above and digested byEcoRI and HindIII.

The preparation of the chromosomal DNA from the cultured tobacco cellswas performed using the Watanabe method (Watanabe, K., Cloning andSequence, Plant Biotechnology Experiment Manual, Nouson Bunka Co.,Ltd.). First, 10 ml of the cultivated tobacco cells were frozen usingliquid nitrogen, and then ground to powder using a mortar and pestle.About five grams of the powder was placed in a centrifuge tube (40 ml)rapidly such that the frozen powder did not melt and mixed with 5 ml ofa 2.times.CTAB (cetyltrimethyl ammonium bromide) solution pre-heated to60° C. This was well mixed, slowly for 10 minutes, and then allowed tostand at 60° C. Then, 5 ml of a chloroform:isoamylalcohol (24:1)solution was added, and the mixture was stirred into and emulsion. Themixture was then centrifuged (2,800 rpm, 15 minutes, room temperature).The surface layer was then transferred to a new 40 ml centrifuge tubeand the extraction process was repeated using thechloroform:isoamylalcohol (24:1) solution. After the surface layer hadbeen mixed with 1/10 volume of 10% CTAB, it was centrifuged (2,800 rpm,15 minutes, room temperature). The surface layer was transferred to anew centrifuge tube and then mixed with an equal volume of coldisopropanol. The thus obtained solvent mixture was then centrifuged(4,500 rpm, 20 minutes, room temperature). After the supernatant hadbeen removed using an aspirator, it was added to 5 ml of a TE buffersolution containing 1 M sodium chloride. This was completely dissolvedat 55-60° C. This was mixed thoroughly with 5 ml of frozen isopropanoland the DNA was observed. It was placed on the tip of a chip,transferred to an EPPENDORF tube (containing 80% frozen ethanol), andthen rinsed. The DNA was then rinsed in 70% ethanol and dried. The driedpellet was dissolved in the appropriate amount of TE buffer solution.Then, 5 ml of RNAase A (10 mg/ml) was added, and reacted for one hour at37° C.; Composition of the 2×CTAB Solution: 2% CTAB, 0.1 M Tris-HCl(pH8.0), 1.4 M sodium chloride, 1% polyvinylpyrrolidone (PVP);composition of the 10% CTAB solution: 10% CTAB, 0.7 M sodium chloride.

The Southern blot method was performed in the following manner:

(i) DNA Electrophoresis and Alkali Denaturation: After 40 μg of thechromosomal DNA had been completely digested by the restriction enzyme,the standard method was used, and 1.5% agarose gel electrophoresis wasperformed (50 V). It was then stained with ethidium bromide andphotographed. The gel was then shaken for 20 minutes in 400 ml of 0.25 MHCl, and the liquid removed, and the gel permeated with 400 ml of adenaturing solution (1.5 M NaCl, 0.5 M NaOH by shaking slowly for 45minutes. Next, the liquid was removed, 400 ml of neutral solution (1.5 MNaCl, 0.5 M Tris-HCl pH 7.4) was added, and the solution was shakenslowly for 15 minutes. Then, 400 ml of the neutral solution was againadded, and the solution was shaken slowly again for 15 minutes. (ii)Transfer: After electrophoresis, the DNA was transferred to a nylonmembrane (Hybond-N Amersham) using 20×SSC. The transfer took more than12 hours. After the blotted membrane was allowed to dry at roomtemperature for an hour, UV fixing was performed for five minutes.20×SSC Composition: 3 M NaCl, 0.3 M sodium citrate. (iii) DNA ProbePreparation: The DNA probe preparation was performed using a RandomPrime Labeling Kit (Takara Shuzo Co., Ltd.). Next, the reaction solutionwas prepared in an Eppendorf tube. After the tube was heated for threeminutes to 95° C., it was rapidly cooled in ice. Then, 25 ng of thetemplate DNA and 2 μl of the Random Primer were added to make 5 μl.Then, 2.5 μl 10× buffer solution, 2.5 μml dNTPs, and 5 μl [α-³²P] dCTP(1.85 MBq, 50 mCi) were added, and H₂O was added to bring the volume ofreaction mixture to 24 μl. Then, 1 μl of a Klenow fragment was added andthe solution was allowed to stand for 10 minutes at 37° C. It was thenpassed through a NAP10 column (Pharmacia Co., Ltd.) to prepare thepurified DNA. After being heated for three minutes at 95° C., it wasrapidly cooled in ice, and used as a hybridization probe. (iv)Hybridization: 0.5% (w/v) SDS was added to the followingPre-hybridization Solution, the membrane in (ii) was immersed in thesolution, and pre-hybridization was performed for more than two hours at42° C. Afterwards, the DNA probe prepared in (iii) was added, andhybridization was performed for more than 12 hours at 42° C. Compositionof the Pre-hybridization Solution: 5×SSC, 50 mM sodium phosphate, 50%(w/v) formamide, 5×Denhardt's solution (prepared by diluting100×Denhardt's solution), 0.1% (w/v) SDS. Composition of the100×Denhardt's Solution: 2% (w/v) BSA, 2% (w/v) Ficol 400, 2% (w/v)polyvinylpyrrolidone (PVP). (v) Autoradiography: After rinsing in themanner described below, autoradiography was performed using the standardmethod. It was performed twice for 15 minutes at 65° C. in 2×SSC and0.1% SDS, and once for 15 minutes at 65° C. in 0.1×SSC and 0.1% SDS.

The results of the Southern blot analysis of the genome DNA preparedfrom the resistant strains are shown in FIG. 4. As shown in FIG. 4, thepresence of the hGT gene was verified in four strains (GT1, 6, 8 and 9).

Example 3 Analysis of the Galactosyltransferase Transformant

The cells of the transformants (GT-1, 6, 8 and 9) and wild-type BY-2 inthe fifth through seventh day's culture both were harvested, and thensuspended in extraction buffer solution (25 mM Tris-HCl, pH 7.4; 0.25 Msucrose, 1 mM MgCl₂, 50 mM KCl). The cells were ruptured usingultrasound processing (200 W; Kaijo Denki Co., Ltd. Japan) orhomogenized. The cell extract solution and the microsome fractions werethen prepared according to the method of Schwientek, T. et al.(Schwientek, T. and Ernst, J. F., Gene 145, 299-303, 1994). Theexpression of the hGT proteins was detected using Western blotting andanti-human galactosyltransferase (GT) monoclonal antibodies (MAb 8628;1:5000) (Uejima, T. et al., Cancer Res., 52, 6158-6163, 1992; Uemura, M.et al., Cancer Res., 52, 6153-6157, 1992) (provided by ProfessorNarimatsu Hisashi of Soka University). Next, the blots were incubatedwith horseradish peroxidase-conjugated goat anti-mouse IgG (5% skim milk1:1000; EY Laboratories, Inc., CA), and a colorimetric reaction usinghorseradish peroxidase was performed using the POD Immunoblotting Kit(Wako Chemicals, Osaka).

An immunoblot analysis of the complex glycans unique to plants wasperformed using polyclonal antiserum against β-fructosidase in the cellwalls of carrots and horseradish peroxidase-conjugated goat anti-rabbitIgG antibodies (5% skim milk 1:1000; Sigma) (Lauriere, M. et al., PlantPhysiol. 90, 1182-1188, 1989).

The β1,4-galactosyltransferase activity was assayed as a substrate usingUDP-galactose and a pyridylamino (PA-) labeled GlcNAc₂Man₃GlcNAc₂(GlcNAc₂Man₃GlcNAc₂-PA) (Morita, N. et al., J. Biochem. 103, 332-335,1988). The enzyme reaction solution contained 1-120 μg protein, 25 mMsodium cacodylate (pH 7.4), 10 mM MnCl₂, 200 mM UDP-galactose, and 100nM GlcNAc₂Man₃GlcNAc₂-PA. An HPLC analysis was performed on the reactionproduct using PALPAK Type R and PALPAK Type N columns (Takara Shuzo Co.,Ltd.) and the method recommended by the manufacturer. TheGlcNAc₂Man₃GlcNAc₂-PA used as the standard marker was used along withGal₂GlcNAc₂Man₃GlcNac₂-PA and two isomers of GalGlcNAc₂Man₃GlcNAc₂-PApurchased from Takara Shuzo Co. Ltd. and Honen Co., Ltd.

The immunoblottings for the proteins derived from the transformant andthe wild-type cells are shown in FIG. 5. As shown in FIG. 5, positivesignals of a molecular weight of 50 kDa were observed. This is greaterthan the molecular weight estimated from the amino acid sequence (40kDa) and is roughly equivalent to the bovine galactosyltransferasepurified from ascites and expressed in yeast (Uemura, M. et al., CancerRes., 52, 6153-6157, 1992; Schwientek, T. et al., J. Biol. Chem., 271(7), 3398-3405, 1996). In the microsome fraction, immunoreactive bands(FIG. 5, Lanes 1, 4) stronger than those of the cell lysate (FIG. 5,Lanes 6-8) were observed. This means that hGT is localizedpreferentially in the cell. No immunoreactive bands were detected in thewild-type cells.

The proteins in the microsome fractions of transformant GT6 andwild-type BY-2 were bound in an RCA₁₂₀ agarose column (Wako Chemicals,Osaka), and then rinsed with 15 volumes of 10 mM ammonium acetate pH6.0. Next, the bound proteins were eluted using 0.2 M lactose. Afterseparation using SDS-PAGE, the proteins were stained using silverstaining (Wako Silver Staining Kit) (FIG. 6) or lectin (FIG. 7). In thelectin staining, the membrane blots were rinsed in a TTBS buffersolution (10 mM Tris-HCl, pH 7.4; 0.15 M NaCl; 0.05% Tween 20) andincubated with horseradish peroxidase labeled RCA₁₂₀ (Honen Co., Ltd.).Galactosylated glycan was then observed using a Immunoblotting Kit (WakoChemicals, Osaka) (FIG. 7). As shown in FIG. 7, an RCA₁₂₀ binding wasnot observed in the wild-type BY2 cells, and the GT6 had a glycoproteinwith galactose on the non-reducing terminus of the glycan portion.

The protein extract from the wild-type BY2 cells and the GT6 cells aswell as the GT6 proteins eluted from the RCA₁₂₀ affinity chromatographywere probed using polyclonal antibodies unique to complex glycan (FIG.8). The antiserum binds predominantly to the β1,2-xylose residue on theplant glycoprotein (Lauriere, M. et al., Plant Physiol. 90, 1182-1188,1989). As shown in FIG. 8, the wild-type BY2 cells (Lane 1) containglycoproteins that reacted with the polyclonal antiserum. GT6 containsvery few glycoproteins that reacted with the polyclonal antiserum (Lane2). The GT6 glycoproteins eluted from RCA₁₂₀ affinity chromatography didnot bind to the polyclonal antiserum, indicating that the galactosylatedglycan does not contain β1,2-xylose residue (Lane 3).

Example 4 Introduction of the Horseradish Peroxidase (HRP) Gene to thehGT-Introduced Cultivated Tobacco Cells

Horseradish peroxidase gene was introduced to the resultant GT6 cellline. Among the different types of plant peroxidase, horseradishperoxidase, especially HRP isozyme C, HRP (EC1.11.1.7) has been thesubject of extensive research. HRP can be used in various enzymereactions because of its superior stability and a broad spectrum ofsubstance specificity. For example, it has been used in enzymeimmunology for binding with a secondary antibody in Western blotting. Anumber of horseradish peroxidase isozyme genes have now been cloned(Fujiyama, K. et al., Eur. J. Biochem., 173, 681-687, 1988 and Fujiyama,K. et al., Gene, 89, 163-169, 1990). ClaPeroxidase (ClaPRX) which isencoded by prxCla is first translated as a protein consisting of 353amino acids containing an extra peptide consisting of 30 amino acids atthe N terminus and 15 amino acids at the C terminus. Then, this isprocessed to form a mature enzyme with 308 amino acids (Fujiyama, K. etal., Eur. J. Biochem., 173, 681-687, 1988). The molecular weight ofClaPRX ranges between 42,200 and 44,000. Of this molecular weight, sugarchains account for 22-27%, and there are eight N-linked sugar chains.(Welinder, K. G., Eur. J. Biochem., 96, 483-502, 1979). The introductionof the ClaPRX gene was performed using the binary vector pBIHm-HRP forHRP expression shown in FIG. 9.

The pBIHm-HRP was prepared in the following manner. First, a 1.9 kbpHindIII-SacI fragment was prepared from a vector 35S-prxCla for plantexpression, which caries an HRP cDNA (Kawaoka, A. et al., J. Ferment.Bioeng., 78, 49-53, 1994).

The HindIII-SacI fragment contains a full length 1.1 kbp prxCla cDNAfollowing a 0.8 kbp CaMV35S promoter. The 1.9 kbp HindIII-SacI fragmentwas inserted in the HindIII-SacI site of the binary vector pBI101HmB(Akama, K. et al., Plant Cell Rep., 12, 7-11, 1992). The BamHI site at3′ of the hygromycin resistant gene (HPT gene) had been destroyed.

Because the GT6 strain is kanamycin resistant, the hygromycin-resistanthpt gene was used as the selection marker (Griz, L. and Davies J., Gene,25, 179-188, 1983). The transformation of the GT6 strain by HRP gene wasperformed using the method described in Rempel, D. H. and Nelson, L. M.(Rempel, D. H. and Nelson, L. M., Transgenic Res. 4: 199-207, 1995). Inorder to obtain HRP transformant as a control, an HRP gene wasintroduced to a wild-type BY2 cell to obtain a BY2-HRP strain. Thedouble-transformant GT6-HRP with hGT and HRP was obtained in which anordinary transformation process takes place.

Example 5 Verification of the Expression of HRP in the CultivatedDouble-Transformant Tobacco Cells

Double transformant GT6-HRP, control BY2-HRP and wild-type (WT) cellline were examined for the expression of HRP activity using thefollowing method. As seen in Table 1, the HRP gene-introducedtransformant had peroxidase activity about five times higher than thewild-type cell line.

TABLE 1 Specific activity Clone Number [U/mg protein] WT-HRP-1 10.3WT-HRP-5 11.3 WT-HRP-7 12.6 GT-HRP-4 11.1 GT-HRP-5 9.35 GT-HRP-8 9.47Wild Type 2.49

Clone BY2-HRP obtained by introducing the HRP gene to the wild typeexpressed the same degree of peroxidase activity as the GT6-HRP doubletransformant with hGT and HRP.

(Peroxidase Activity Measurement)

The cultivated tobacco cells were placed into an Eppendorf tubecontaining Solution D and were ruptured using a homogenizer (HomogenizerS-203, Ikeda Rika Co., Ltd.). The supernatant was collected aftercentrifugation (12,000 rpm, 20 minutes, 4° C.) and then used as thecrude enzyme solution. Next, 1 ml of Solution A, 1 ml of Solution B and2 ml of Solution C were mixed together, and the mixture was incubatedfor five minutes at 25° C. The crude enzyme solution appropriatelydiluted with Solution D was added to the mixture, and allowed to reactfor three minutes at 25° C. The reaction was stopped by the addition of0.5 ml of 1 N HCl, and the absorbance at 480 nm was measured. As acontrol, a solution with 1 N HCl added before the introduction of theenzyme was used.

Solution A: 1 mM o-aminophenol

Solution B: 4 mM H₂O₂

Solution C: 200 mM sodium phosphate buffer (pH 7.0)

Solution D: 10 mM sodium phosphate buffer (pH 6.0)

Next, in order to determine whether or not the rise in peroxidaseactivity was due to the expression of HRP, activity staining wasperformed after separation by gel isoelectric focusing. The isoelectricfocusing was performed using a BIO-RAD Model 111 Mini-IEF Cell. Thehydrophobic surface of the PAGE gel support film was attached to a glassplate, and then placed on a casting tray. The prepared gel solution waspoured between the support film and the casting tray and thenphotopolymerized for 45 minutes under a fluorescent lamp. The sample wasapplied to the gel, and the gel was positioned so as to come intocontact with both graphite electrodes wetted with distilled water in theelectrophoretic bath. Electrophoresis was then performed for 15 minutesat 100 V, 15 minutes at 200 V and 60 minutes at 450 V. Composition ofthe Gel Solution (per 1 Gel Sheet):distilled water 2.75 ml, acrylamide(25% T, 3% C) 1.0 ml, 25% glycerol 1.0 ml, Bio-lite (40%, pH 3-10) 0.25ml, 10% ammonium persulfate 7.5 μl, 0.1% sodium riboflavin5′-phosphate25 μl, TEMED 1.5 μl.

The activity staining of peroxidase was performed according to themethod of Sekine et al. (Sekine et al., Plant Cell Technology, 6, 71-75,1994). As shown in FIG. 10, a significant band not found in wild-typecell line was detected in the pI 7.8 position in the BY2-HRP cell lineand the GT6-HRP strain. The results of a Western analysis using anti-HRPantibodies confirmed the detection of a signal at the positioncorresponding to pI 7.8 and the expression of HRP in the doubletransformant GT6-HRP with hGT and HRP.

Example 6 Structural Analysis of the N-Linked Sugar Chains in theTransformant GT6 Cells

(Method Used to Analyze the Sugar Chain Structure)

The N-linked sugar chains in the transformant GT6 cells were analyzed bycombining reverse-phase HPLC and size-fractionation HPLC, performing thetwo-dimensional PA sugar chain mapping, performing exoglycosidasedigestion, and then performing ion spray tandem mass spectrometry(IS-MS/MS) (Perkin Elmer Co., Ltd.). First, the cell extract solutionwas delipidated with acetone, treated with hydrazine for 12 hours at100.degree. C., and the sugar chain portion was released. Thehydrazinolysate was N-acetylated, desalted using the DOWEX 50×2 and theDOWEX 1×2 (The Dow Chemical Co., Ltd. and its representative in Japan,Muromachi Chemical Industry Co., Ltd.), then fractionized by using 0.1 Nammonia and the SEPHADEX G-25 gel filtration column (1.8.times.180 cm)(Pharmacia Co., Ltd.). Pyridylamination was then performed as describedabove. The pyridylaminated sugar chains (PA sugar chains) were thenseparated using a JASCO 880-PU HPLC device with a JASCO 821-PPIntelligent Spectrophotometer (Japan Spectroscopic Co., Ltd.) andCOSMOSIL 5C18-P and ASAHIPAK NH2P-50 columns. The elution positions werecompared with a standard either produced by the applicant or purchased(from Wako Pure Chemical Industries, Ltd. and Takara Shuzo Co., Ltd.).

The glycosidase digestion using N-acetyl-β-D-glucosaminidase(Diplococcus pneumoniae, Boehringer Mannheim) or mannosidases (Jackbean, Sigma) was performed on about 1 nmol of the PA sugar chains underthe same conditions as the method described in Kimura, Y. et al.,Biosci. Biotech. Biochem. 56 (2), 215-222, 1992. Digestion usingβ-galactosidase (Diplococcus pneumoniae, Boehringer Mannheim) orAspergillus saitoi-derived α-1,2 mannosidase (provided by Dr. TakashiYoshida at Tohoku University) was performed by adding 1 nmol of PA sugarchains and 200 mU β-galactosidase or 60 μg of α-1,2 mannosidase to 50 mMof sodium acetate buffer (pH 5.5) and incubating at 37° C. After theresultant reaction solution was boiled and the enzyme reaction wasstopped, a portion of the digested product was analyzed usingsize-fractionation HPLC. The molecular weight of the digested productwas analyzed using ion spray tandem mass spectrometry (IS-MS/MS) and/orcompared to the standard sugar chain as described in Palacpac, N. Q. etal., Biosci. Biotech. Biochem. 63 (1) 35-39, 1999 and Kimura, Y. et al.,Biosci. Biotech. Biochem. 56 (2), 215-222, 1992.

The IS-MS/MS experiment was performed using a Perkin Elmer SciexAPI-III. It was performed in positive mode with an ion spray voltage of4200 V. Scanning was performed every 0.5 Da, and the m/z was recordedfrom 200.

(Analysis of the Sugar Chains in the GT6 Cells)

The PA sugar chains prepared from the GT6 cells were purified andanalyzed using a combination of reverse-phase HPLC andsize-fractionation HPLC. In Fraction I at the 10-20 minute positions inthe size-fractionation HPLC (FIG. 11), no N-linked sugar chains wereeluted. This suggests that the Fraction I is a non-absorption portioncontaining by-products of hydrazinolysis. In the MS/MS analysis, nofragment ion with m/z values of 300, which corresponds to PA-GlcNAc, wasdetected. Similarly, Fraction XI at the 50-60 minute positions did nothave a peak indicating elution by the size-fractionation HPLC.Therefore, it is clear that there were no N-linked sugar chains. The 17peaks including A-Q shown in FIG. 12 were all collected and purifiedafter the analysis from Fraction II to Fraction X in thesize-fractionation HPLC (FIG. 11) was completed.

The IS-MS/MS analysis found that seven of these peaks were N-linkedsugar chains. The following is the result from the analysis of thesepeaks.

The elution positions and molecular weights of oligosaccharides-A, -E,-H, -I, -M, -O, -P and -Q (FIG. 12) did not correspond to those of PAsugar chain standards. In the MS/MS analysis, the m/z values of 300 and503, which respectively correspond to PA-GlcNAc and PA-GlcNac₂, weredetected. However, the fragment ions were not detected corresponding toManGlcNA₂ (M1) or the trimannose core sugar chain Man₃GlcNAc₂ (M3) whichare generally found in N-linked sugar chain (data not shown). Even theoligosaccharides-B, -D and -N at the other peaks did not have fragmentions detected with an m/z value of 300. Thus, these were not N-linkedsugar chains. The seven remaining N-linked sugar chains were thenexamined.

The elution positions and molecular weights of peak-C (m/z 1637.5; molarratio 9.3%), peak-F ([M+2H] 2+m/z 819.5, [M+H]+m/z 1639; molar ratio15.9%), and peak-G (m/z 1475.5; molar ratio 19.5%) indicated highmannose-type sugar chains Man₇GlcNAC₂ (Isomer M7A and M7B) andMan₆GlcNAc₂ (M6B) respectively. When digested by Jack beanα-mannosidase, it was indicated that the N-linked sugar chains aredegraded to ManGlcNAc (M1) by size-fractionation HPLC analysis (data notshown). In an IS-MS experiment on the digestion product, the ion with anm/z value of 665.5 corresponding to a calculated value of 664.66 for M1was detected, indicating that these N-linked sugar chains have the samestructure as respective corresponding PA sugar chain standard.

Peak-J (6.6%) had a molecular weight of 1121.5, which is almost the sameas the calculated molecular weight value of m/z 1121.05 ofMan₃Xyl₁GlcNAc₂-PA (M3X). The positions of the fragment ions were 989.5,827.5, 665.5, 503.3 and 300. This does not contradict the findings thatXyl, Man, Man, Man, and GlcNAc were released in successive order fromMan₃Xyl₁GlcNAc₂-PA. When digested using Jack bean α-mannosidase, themannose residue on the non-reducing terminus can be removed, and thetwo-dimensional mapping revealed the same elution positions as those ofMan₁Xyl₁GlcNAc₂-PA (data not shown).

The results of the analysis of the IS-MS experiment on peak-K (13.2%)fraction revealed that this fraction contains two types of N-linkedsugar chains, one has the molecular weight of 1314.0 (1.4%) and theother has the molecular weight of 1354.5 (11.8%). This fraction wassubjected to reverse-phase HPLC, purified and analyzed. The sugar chainpeak K-1 with a molecular weight of 1314.0 had the same two-dimensionalmapping and m/z value measured as that of the sugar chain standardMan₅GlcNAc₂-PA (M5). When treated using jack bean α-mannosidase, theelution positions of the degradated product had shifted to positionssimilar to those of M1 in the two-dimensional mapping. This indicatesthe removal of four mannose residues.

(Galactose-Added N-Linked Type Sugar Chains in the GT6 Cells)

The determined m/z value of 1354.5 for sugar chain peak K-2 is almostthe same as the molecular weight m/z value of 1354.3 predicted forGal₁GlcNAc₁Man₂GlcNAc₂-PA (GalGNM3). The result of the mass spectrometryindicated that fragment ions were derived from the parent molecules. Them/z value of 1193.5 indicated GlcNAc₁Man₃GlcNAc₂-PA, the m/z value of989.5 indicated Man₃GlcNAc₂-PA, the m/z value of 827.5 indicatedMan₂GlcNAc₂-PA, the m/z value of 665 indicated ManGlcNAc₂-PA, the m/zvalue of 503 indicated GlcNAc₂-PA, the m/z value of 336 indicatedManGlcNAc, the m/z value of 300 indicated GlcNAc-PA, and the m/z valueof 204 indicated GlcNAc. From the putative N-linked sugar chainstructure, it is considered to be either of two GalGNM3 isomers (FIG.13). It is either Gal β4GlcNAc β2Man α6 (Man 3) Man β 4GlcNacβ4GlcNAc-PA or Man α6 (Gal β4GlcNAc β2Man α3) Man β 4GlcNAc β4GlcNAc-PA.The purified PA sugar chains had reverse-phase HPLC elution positionsthat were the same as the sugar chain standard Man α6 (Gal β4GlcNAcβ2Man α3) Man β4GlcNAc β4GlcNAc-PA (FIG. 13B).

The sugar chain was treated with exoglycosidase and the structure of thesugar chain was verified. The D. pneumoniae β-galactosidase is a Galβ1,4GlcNAc linkage specific enzyme. The digested product of the sugar chainby the enzyme was eluted at the same position as that of theGlcNAc₁Man₃GlcNAc₂-PA in the size-fractionation HPLC (FIG. 14A-II). Anm/z of 1192.0 was obtained from the IS-MS/MS analysis. These resultsindicate a galactose residue has been removed from the GlcNAc on thenon-reducing terminus with the β1,4 binding. When the product wasdigested by a N-acetyl-β-D-glucosaminidase derived from Diplococcuspneumoniae, which is β1,2GlcNAc linkage specific (Yamashita, K. et al.,J. Biochem. 93, 135-147, 1983), the digested product was eluted at thesame position as that of the standard Man₃GlcNAc₂-PA in thesize-fractionation HPLC (FIG. 14A-III). When the digested product wastreated with jack bean α-mannosidase, it was eluted at the same positionas that of the standard ManGlcNAc₂-PA in the size-fractionation HPLC(FIG. 14A-IV). The sugar chain structure is shown in K-2 of FIG. 15.

The mass spectroscopy analysis of Peak L (35.5%) gave [M+2H] 2+ of 840,[M+H]+ of 1680.0, which nearly matched the molecular weight m/z value of1678.55 expected for Gal₁GlcNAc₁Man₅GlcNAc₂-PA (GalGNM5). The result ofthe mass spectrometry indicated fragment ions derived from the parentmolecules. The m/z value of 1313.5 indicated Man₅GlcNAc₂-PA, the m/zvalue of 1152 indicated Man₄GlcNAc₂-PA, the m/z value of 989.5 indicatedMan₃GlcNAc₂-PA, the m/z value of 827.5 indicated Man₂GlcNAc₂-PA, the m/zvalue of 665 indicated ManGlcNAc₂-PA, the m/z value of 503 indicatedGlcNAc₂-PA, the m/z value of 336 indicated ManGlcNAc, the m/z value of300 indicated GlcNAc-PA, and the m/z value of 204 indicated GlcNAc. Theproduct digested with D. pneumoniae β-galactosidase was eluted at thesame position as that of GlcNAc₁Man₅GlcNAc₂-PA in the size-fractionationHPLC (FIG. 14B-II). The results indicate that a galactose residue isbound to the GlcNAc on the non-reducing terminus with the β1,4 linkage.The removal of the galactose was confirmed by the molecular weightsobtained from the IS-MS/MS analysis. [M+2H] 2+ was 759 and [M+H] was1518.0. The mass spectrometry indicated fragments ions derived from theGlcNAc₁Man₅GlcNAc₂-PA with a parent signal of m/z 1518.0. The m/z valueof 1314 indicated Man₅GlcNAc₂-PA, the m/z value of 1152 indicatedMan₄GlcNAc₂-PA, the m/z value of 990 indicated Man₃GlcNAc₂-PA, the m/zvalue of 827.5 indicated Man₂GlcNAc₂-PA, the m/z value of 665.5indicated Man₁GlcNAc_(z)-PA, the m/z value of 503 indicated GlcNAc₂-PA,and the m/z value of 300 indicated GlcNAc-PA. When theGlcNAc₁Man₅GlcNAc₂-PA was digested with an N-acetyl-β-D-glucosaminidasederived from Diplococcus pneumoniae, the digested product was eluted atthe same position as that of the standard Man₅GlcNAc₂-PA in thesize-fractionation HPLC (FIG. 14B-III). Even when treated with α-1,2mannosidase derived from Aspergillus saitoi, the elution position didnot shift (FIG. 14B-IV). However, when treated with jack beanα-mannosidase, it was eluted at the same position as that of standardMan₁GlcNAc₂-PA in the size-fractionation HPLC (FIG. 14B-V). Thisindicates the removal of four mannose residues in the non-reducingterminus. These results indicate that in the PA sugar chain, none offive mannose residues are α1, 2 linked to the mannose residue which areα1,3 binding. The exoglycosidase digestion, two-dimensional sugar chainmapping, and IS-MS/MS analysis indicate a sugar chain structure ofGalGNM5 as shown by L in FIG. 15.

FIG. 20 summarizes the above results regarding the structure of N-linkedglycans and the ratio of each N-linked glycan in GT6 cell line alongwith those in wild-type BY2 cell line determined similarly. In FIG. 20,□ denotes GlcNAc, ◯ denotes mannose, ● denotes galactose, □ with hatchedlines therein denotes xylose, and ◯ with dots therein denotes fucoserespectively.

In GT6 cell line, the isomers Man7-, Man6- and Man5GlcNAc2 wereobserved. Because those high-mannose type oligosaccharides will besubstrates for β1,4-galactosyltransferase (Gal T), introduction ofGlcNAc I, Man I and Man II cDNAs can more efficiently lead theoligosaccharide Man7-5GlcNAc2 to GlcNAcMan3GlcNAc2, which can be asubstrate of GalT (FIG. 21).

A. thaliana cglI mutant, that lacks GnT I, can not synthesize complextype N-glycans (von Schaewen, A., Sturm, A., O'Neill, J., andChrispeels, M J., Plant Physiol., 1993 August; 102 (4):1109-1118,Isolation of a mutant Arabidopsis plant that lacks N-acetyl glucosaminyltransferase I and is unable to synthesize Golgi-modified complexN-linked glycans). Complementation with the human GnT I in the cglImutant indicated that the mammalian enzyme could contribute the plantN-glycosylation pathway (Gomez, L. and Chrispeels, M. J., Proc. Natl.Acad. Sci. USA 1994 Mar. 1; 91 (5):1829-1833, Complementation of anArabidopsis thaliana mutant that lacks complex asparagine-linked glycanswith the human cDNA encoding N-acetylglucosaminyltransferase I.)Furthermore, GnT I cDNA isolated from A. thaliana complementedN-acetylglucosaminyltransferase I deficiency of CHO Lec1 cells (Bakker,H., Lommen, A., Jordi, W., Stiekema, W., and Bosch, D., Biochem.Biophys. Res. Commun., 1999 Aug. 11; 261 (3):829-32, An Arabidopsisthaliana cDNA complements the N-acetylglucosaminyltransferase Ideficiency of CHO Lec1 cells). cDNAs encoding human Man I and Man IIwere isolated and sequenced (Bause, E., Bieberich, E., Rolfs, A.,Volker, C. and Schmidt, B., Eur J Biochem 1993 Oct. 15; 217 (2):535-40,Molecular cloning and primary structure of Man9-mannosidase from humankidney; Tremblay, L. O., Campbell, Dyke, N. and Herscovics, A.,Glycobiology 1998 June; 8 (6):585-95, Molecular cloning, chromosomalmapping and tissue-specific expression of a novel human alpha1,2-mannosidase gene involved in N-glycan maturation; and Misago. M.,Liao, Y. F., Kudo, S., Eto, S., Mattei, M. G., Moremen, K. W., Fukuda,M. N., Molecular cloning and expression of cDNAs encodinghumanalpha-mannosidase II and a previously unrecognizedalpha-mannosidase IIx isozyme). Human Man I has two isozymes, Man IA andMan IB, and the nucleotide structure of isozymes' cDNA was shown (Bause,E., et al., and Tremblay, L. O., supra).

By transforming these cDNAs into the BY cell line, an efficient cellline producing human-type glycoprotein, can be obtained.β1,4-Galactosyltransferase (Gal T) uses UDP-galactose as a donorsubstrate and GlcNAc2Man3GlcNAc2 as an acceptor substrate. Efficientsupply of UDP-galactose will enhance the Gal T enzyme reaction, and moregalactosylated oligosaccharide will be produced (FIG. 22).

Example 7 Structural Analysis of the Sugar Chains on the HRP in theDouble Transformant GT6-HRP Cells

A crude cell lysate was obtained from the homogenate of 50 g of culturedGT6-HRP cells or control BY2-HRP cells grown for seven days,respectively. This crude cell lysate solution was applied to a CMSEPHAROSE FF column (1×10 cm) (Pharmacia Co., Ltd.) equilibrated with 10mM of sodium phosphate buffer (pH 6.0). After washing the column, theeluted peroxidase was measured at an absorbance of 403 nm. The pooledfraction was concentrated using an ultrafilter (molecular weight cutoff: 10,000, Advantec Co., Ltd.), dialyzed against 50 mM of a sodiumphosphate buffer (pH 7.0), and then applied to an equilibratedbenzhydroxaminic acid-agarose affinity column (1×10 cm) (KemEn Tech,Denmark). After the column was washed in 15 volumes of 50 mM of sodiumphosphate buffer (pH 7.0), the absorbed HRP was eluted using 0.5 M boricacid prepared in the same buffer. The peroxidase active fractionobtained was then pooled, dialyzed, and concentrated.

The purified HRP prepared from the double transformant GT6-HRP cells orBY2-HRP cells was applied to a 1×10 cm RCA₁₂₀-agarose column. The columnwas then washed with 15 volumes of 10 mM ammonium acetate (pH 6.0). Theabsorbed proteins were then eluted and assayed using conventionalmethods.

Lectin staining was then performed on the purified HRP eluted fromRCA₁₂₀ affinity chromatography whose specificity is specific to β1,4linkage galactose. The lectin RCA₁₂₀ was bound to only the HRP producedby the transformed cell GT6-HRP. Because the lectin binding wasdramatically reduced by preincubation with the galactose which cancompete with the lectin (FIG. 16 b-III), the binding is carbohydratespecific. Even when the purified HRP is pre-treated with D. pneumoniaeβ-galactosidase, the RCA₁₂₀ binding was inhibited. These resultsindicate RCA bound specifically to β1,4-linked galactose at thenon-reducing end of N-linked glycan on HRP. The absence of RCA-boundglycoproteins in the BY2-HRP cells indicates that these cells can nottransfer the β1,4 linked galactose residue to the non-reducing terminusof the HRP glycan.

Reverse-phase HPLC of PA derivatives derived from HRP purified usingRCA₁₂₀ indicated that the sugar chains on the HRP proteins purified fromthe GT6-HRP appear as a single peak (FIG. 17). In the reverse phaseHPLC, a Cosmosil 5C18-P column or ASAHIPAK NH2P column was used in aJASCO 880-PU HPLC device with a JASCO 821-FP IntelligentSpectrofluorometer. Neither bound proteins nor detectable peaks wereobserved in the HRP fractions purified from BY2-HRP. The peak obtainedfrom the GT6-HRP in the size-fractionation chromatography washomogenous. The two-dimensional mapping analysis of the peak andchromatography of the peak at the same time with standard sugar chainindicated that the oligosaccharide contained in the peak wasGal₁GlcNAc₁Man₅GlcNAc₂-PA. The confirmation of this structure wasprovided using continuous exoglycosidase digestion. The standard sugarchains used were a sugar chains prepared previously (Kimura, Y. et al.,Biosci. Biotech. Biochem. 56 (2), 215-222, 1992) or purchased (Wako PureChemical, Industries, Ltd. Osaka and Takara Shuzo Co., Ltd.).

The PA sugar chain digested with β-galactosidase (D. pneumoniae) matchedthe elution position of the standard GlcNAc₁Man₅GlcNAc₂-PA indicatingthe removal of a galactose residue β1,4-linked to a non-reducingterminal GlcNAc. Further digestion with D. pneumoniaeN-acetyl-β-D-glucosaminidase of β-galactosidase-digested productsproduced a sugar chain equivalent which is eluted at the same elutionposition of Man₅GlcNAc₂-PA, indicating the removal of a GlcNAc residueβ1,2 linked to a non-reducing terminal mannose residue. The removedGlcNAc residue is believed to be linked to α1,3 mannose linked to a β1,4mannose residue in view of the N-linked type processing route of theplant. In order to confirm the linkage position of the GlcNAc residue,Man₅GlcNAc₂-PA (M5) was incubated with α1,2 mannosidase derived fromAspergillus saitoi. As expected, an elution position shift was notdetected, confirming M5 has the structure Man α1-6 (Man α1,3) Man α 1-6(Man α1,3) Man β1,4GlcNAc β1,4GlcNAc as predicted. When the sugar chainwas digested using jack bean α-mannosidase, it was eluted at the sameelution positions of known Man₁GlcNAc₂-PA. Therefore, the sugar chainstructure corresponded to Man α1-6 (Man α1,3) Man α1-6 (Gal β1, 4GlcNAcβ1, 2Man α1,3) Man β1,4GlcNAc β1,4GlcNAc (Gal₁GlcNAc₁Man₅GlcNA₂). Theseresults indicate that the sugar chain in GT6 cell has the structureshown in FIG. 15 and that the sugar chain structure on an HRP proteinderived from the double transformant GT6-HRP is Man α1-6 (Man α 1, 3)Man α1-6 (Gal β1, 4GlcNAc β1, 2Man α1, 3) Man β1, 4GlcNAc β 1,4GlcNAc(Gal₁GlcNAc₁Man₅GlcNA₂).

Similarly, the galactosylated N-glycan on HRP derived from thetransformant GT6-HRP cells did not react with an antiserum which hasbeen shown to specifically react with β1,2 xylose residue indicative ofplant N-glycans. This indicates that one of the sugar residues shown tobe antigenic in complex plant glycan, i.e., xylose residue, is notpresent (Garcia-Casado, G. et al., Glycobiology 6 (4): 471,477, 1996)(FIG. 18).

INDUSTRIAL APPLICABILITY

The present invention provides a method for manufacturing a glycoproteinwith a human-type sugar chain. It also provides plant cells that have asugar chain adding mechanism able to perform a reaction in which agalactose residue is transferred to a acetylglucosamine residue on thenon-reducing terminal, wherein the sugar chain adding mechanism iscapable of joining a sugar chain which contains a core sugar chain andan outer sugar chain, wherein the core sugar chain consists essentiallyof a plurality of mannose and acetylglucosamine, and the outer sugarchain contains a terminal sugar chain portion containing a galactose onthe non-reducing terminal. The present invention further provides aglycoprotein with a human-type sugar chain obtained by the presentinvention. A glycoprotein with a mammalian, e.g., human-type sugar chainof the present invention is not antigenic because the glycosylation is ahuman-type. Therefore, it can be useful for administering to animalsincluding humans.

The invention claimed is:
 1. A plant-produced glycoprotein, comprising(i) a galactosylated sugar chain that comprises a galactose residuelinked to a N-acetylglucosamine residue in β1,4-linkage, and (ii) anon-galactosylated sugar chain comprising a xylose residue linked to amannose residue in β1,2-linkage.
 2. The plant-produced glycoprotein ofclaim 1, wherein no α1,3-fucose, and/or no β1,2-xylose is linked to thegalactosylated sugar chain.
 3. The plant-produced glycoprotein of claim1, wherein the glycoprotein comprises N-glycans of M7A, M7B, M6B, M3X,M5, GalGNM3, and GalGNM5, wherein M is mannose, X is xylose, Gal isgalactose, and N is N-acetylglucosamine, and wherein the structures ofM7A, M7B, M6B, M3X, M5, GalGNM3, and GalGNM5 are set forth in FIG.
 1. 4.The plant-produced glycoprotein of claim 1, wherein the plant-producedglycoprotein is an enzyme, a hormone, a cytokine, an antibody, a vaccineantigen, a receptor, or a serum protein.
 5. The plant-producedglycoprotein of claim 4, wherein the plant-produced glycoprotein is anenzyme selected from the group consisting of horseradish peroxidase,kinase, glucocerebrosidase, α-galactosidase, tissue-type plasminogenactivator (TPA), and 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA)reductase.
 6. The plant-produced glycoprotein of claim 4, wherein theplant-produced glycoprotein is selected from the group consisting ofenkephalin, interferon-alpha, granulocyte-macrophage colony stimulatingfactor (GM-CSF), granulocyte colony stimulating factor (G-CSF), chorionstimulating hormone, interleukin-2, interferon-beta, interferon-gamma,erythropoietin, vascular endothelial growth factor, humanchoriogonadotropin (HCG), leuteinizing hormone (LH), thyroid stimulatinghormone (TSH), prolactin, and ovary stimulating hormone.
 7. Theplant-produced glycoprotein of claim 4, wherein the plant-producedglycoprotein is an antibody.
 8. The plant-produced glycoprotein of claim1, wherein the galactose residue is a terminal sugar residue in thegalactosylated sugar chain.
 9. The plant-produced glycoprotein of claim1, wherein the plant-produced glycoprotein is produced by a processcomprising: cultivating a transformed plant cell comprising a first geneencoding a galactosyltranferase, and a second gene encoding theglycoprotein under conditions allowing expression of thegalactosyltransferase and the glycoprotein; wherein the transformedplant cell further comprises one or more genes encoding one or more ofN-acetylglucosaminyl-transferase I, mannosidase I, and mannosidase II;and isolating the glycoprotein, which comprises a human-type sugar chaincomprising a galactose residue linked to a N-acetylglucosamine residue,wherein addition of the galactose residue is catalyzed by thegalactosyltransferase.
 10. The plant-produced glycoprotein of claim 9,wherein no α1,3-fucose, nor and/or no β1,2-xylose is linked to thegalactosylated sugar chain.
 11. The plant-produced glycoprotein of claim9, wherein the glycoprotein comprises N-glycans of M7A, M7B, M6B, M3X,M5, GalGNM3, and GalGNM5, wherein M is mannose, X is xylose, Gal isgalactose, and N is N-acetylglucosamine, and wherein the structures ofM7A, M7B, M6B, M3X, M5, GalGNM3, and GalGNM5 are set forth in FIG. 15.12. The plant-produced glycoprotein of claim 9, wherein glycoprotein isan enzyme, a hormone, a cytokine, an antibody, a vaccine antigen, areceptor, or a serum protein.
 13. The plant-produced glycoprotein ofclaim 12, wherein the plant-produced glycoprotein is an enzyme selectedfrom the group consisting of horseradish peroxidase, kinase,glucocerebrosidase, α-galactosidase, tissue-type plasminogen activator(TPA), and 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase.14. The plant-produced glycoprotein of claim 12, wherein theplant-produced glycoprotein is selected from the group consisting ofenkephalin, interferon-alpha, granulocyte-macrophage colony stimulatingfactor (GM-CSF), granulocyte colony stimulating factor (C-CSF), chorionstimulating hormone, interleukin-2, interferon-beta, interferon-gamma,erythropoietin, vascular endothelial growth factor, humanchoriogonadotropin (HCG), leuteinizing hormone (LH), thyroid stimulatinghormone (TSH), prolactin, and ovary stimulating hormone.
 15. Theplant-produced glycoprotein of claim 12, wherein the plant-producedglycoprotein is an antibody.
 16. The plant-produced glycoprotein ofclaim 9, wherein the first gene encodes a galactosyltransferasecomprising the amino acid sequence of SEQ ID NO:6.
 17. Theplant-produced glycoprotein of claim 13, wherein the first genecomprises the nucleotide sequence of SEQ ID NO:5.